Figure 3

Madecassic acid restored the T helper type 17/regulatory T cell (Th17/Treg) balance by enhancing the shift of of Th17 toward Treg cells. (a) Naive T cells were differentiated under Th17-inducing conditions for 4 days in the presence of madecassic acid (1, 3, 10 μM) or a control. The proportions of CD4⁺IL-17⁺ T cells and CD4⁺Foxp3⁺ T cells were gated by flow cytometry. (b) Naive T cells were differentiated under Treg-inducing conditions for 4 days in the presence or absence of madecassic acid (1, 3, 10 μM). The proportions of CD4⁺IL-17⁺ T cells and CD4⁺Foxp3⁺ T cells were gated by flow cytometry. (c) Naive T cells were cultured with madecassic acid (1, 3, 10 μM) under Th17-inducing conditions for 4 days. The mRNA levels of IL-17A, IL-17F, IL-21, IL-22 and IL-10 were measured by real-time PCR. (d) Naive T cells were cultured with madecassic acid (1, 3, 10 μM) under Th17-inducing conditions for 4 days. The expression of RORγt and Foxp3 under Th17-polarizing conditions was measured by immunofluorescence. (e) Naive T cells were cultured with madecassic acid (1, 3, 10 μM) under Th17-inducing conditions for 4 days. The protein expression of RORγt and Foxp3, as well as Th17-associated transducers p-AKT, AKT, p-STAT3, STAT3, p-JAK2 and JAK was examined by western blot. (f) Naive T cells were cultured with madecassic acid (1, 3, 10 μM) under Th17-inducing conditions for 4 days. The mRNA expression of RORγt and Foxp3 was analyzed by real-time PCR. GAPDH was used as a cytoplasm marker; Lamin B1 was used as a nuclear marker. The data were expressed as means±S.E.M., n=3. ^#P<0.05, ^##P<0.01 versus Th0 group; *P<0.05, **P<0.01 versus Th17 group